WO2000000598A2 - Plant dwarfing - Google Patents

Abstract

The present invention concerns a novel plant gene whose expression results in a dwarf phenotype, genetic constructs comprising same, plants modified to express same, and containing said constructs, seed obtained by growing said plants, methods of conferring a dwarf phenotype upon plants, plants obtained by said methods, and seed obtained by growing same.

Description

Plant Dwarfing

The present invention concerns a novel plant gene whose expression results in a dwarf phenotype, genetic constructs comprising same, plants modified to express same, and containing said constructs, seed obtained by growing said plants, methods of conferring a dwarf phenotype upon plants, plants obtained by said methods, and seed obtained by growing same.

In horticulture, it is desirable to control the elongation growth of individual plant organs and overall plant stature and architecture. The present inventor has now succeeded in isolating a novel member (named mybntl) of the myb family of plant genes. mybntl normally being predominantly expressed in pollen, but which when expressed in other plant organs induces a dominant dwarf phenotype, the extent of dwarfing being dependent upon the level of expression of mybntl.

The myb genes are a family of pathway regulating genes distinguished by a conserved DNA binding domain (Martin, C. and Paz-Ares, J., 1997, TIG. 11(2): 67- 73). Modified expression of myb family genes has previously been shown to affect pigment synthesis, cell shape and thus colour due to diffraction, root hair shape, timing of flowering, response to hormones during seed development and germination, and lignin synthesis. In the case of affected lignin synthesis (Tamagnone, L. et al., 1998. The Plant Cell, JO: 135-154), dwarfing was observed but it also resulted in premature senescence resulting in necrotic areas in older leaves, and also chlorosis (i.e. reduced chlorophyll content).

The present invention provides the opportunity to produce transgenic plants which express mybntl for example constitutively or in an organ-specific manner, to effect a modification of plant architecture e.g. a general dwarfing, whilst avoiding the aforementioned disadvantages.

According to the present invention there is provided a plant gene having the sequence of SEQ ID NO: 1. Plants expressing the gene of the present invention overcome the prior art disadvantages, having a dwarf phenotype whilst not suffering from the severe chlorosis and early senescence associated with other yb-mediated dwarfing.

Naturally, the coding sequence of SEQ ID NO: 1 may be modified whilst still retaining its dwarfing capabilities. For example, the nucleic acid sequence may be modified yet still encode the same codons i.e. the same expression product. Similarly, it is possible to substitute, delete or add nucleotides to modify the coding sequence expression product. Such modification is a simple task for one skilled in the art using standard techniques and the results of the modifications upon dwarfing may be readily determined.

As discussed above, this gene is predominantly expressed in pollen (see "Experimental" below) and its expression in other plant organs results in dwarfing (see "Experimental" below).

Also provided according to the present invention is an expression product encoded by same. Expression producst of this class of genes are molecules containing DNA binding domains.

The gene may be provided as part of a genetic construct, and thus the present invention also provides a recombinant DNA construct whose expression in plants induces a dwarf phenotype, and comprising operatively linked in sequence in the 5' to 3' direction: a) a promoter region that directs the transcription of a gene in plants;

b) a DNA coding sequence encoding an RNA sequence encoding an expression product according to the present invention; and

c) a 3' non-translated region which contains a polyadenylation signal for plants.

The expression of the construct may be in plant tissues other than pollen.

The DNA coding sequence in such a construct may have the sequence of SEQ ID NO: 1.

The promoter may comprise the CaMV35S promoter. The 3' non-translated region may comprise the 3' polyadenylation signal from the CaMV35S transcript.

Promoters useful in the present invention preferably possess one or more of the following properties: constitutive expression of inserted sequences throughout the plant; intermediate rate of expression of inserted sequences; organ-specific expression in stems and/or petioles and/or leaf sheathes; cell-specific expression in vascular tissues. These properties are defined as to drive expression of the inserted sequence(s) at a level that results in mybntl stimulation to produce dwarfing when the modified plants are grown in the field.

Examples of promoters useful in the present invention include, inter alia, the cauliflower mosaic virus 35S (CaMV 35S) promoter, the maize polyubiquitin (ubl) promoter (Christensen, A.H. et al., 1992, Plant Mol. Biol. 18: 675-689), the Asparagus officinalis pathogenesis-related vascular-tissue-specific (AoPRl) promoter (Warner, S.A.J. et al., 1994, Plant Journal, 6: 31-43) the rice ribulose-bisphosphate carboxylase (rbcs) promoter (Kyozuka, J. et al., Plant Physiology, 102: 991-1000). and the maize shrunken- 1 promoter (Maas, C. et al, Plant Mol. Biol., 16: 199-207).

Also provided according to the present invention are plants transformed or transfected with said genetic construct. Methods of transformation and transfection of both monocotyledenous and dicptyledenous plants are well known.

Methods for producing transgenic plants in a variety of different monocots are currently available, and these methods are equally applicable to the present invention. Successful transformation and plant regeneration have been achieved in asparagus (Asparagus officinalis; Bytebier et al, 1987, Proc. Natl. Acad. Sci. USA 84: 5345); barley (Hordeum vulgarae; Wan and Lemaux, 1994, Plant Physiol. 104: 37); maize (Zea mays; Rhodes et al., 1988, Science 240: 204; Gordon-Kamm et al., 1990, Plant Cell 2: 603; Fromm et al.. 1990, Bio/Technology 8: 833; Koziel et al., 1993, Bio/Technology 11 : 194); oats (Avena sativa; Somers et al, 1992, Bio/Technology 10: 1589); orchardgrass (Dactylis glomerata; Horn et al., 1988, Plant Cell Rep. 7: 469); rice (Oryza sativa, including indica and japonica varieties; Toriyama et al., 1988, Bio/Technology 6: 10; Zhang et al., 1988, Plant Cell Rep. 7: 379; Luo and Wu, 1988, Plant Mol. Biol. Rep. 6: 165; Zhang and Wu, 1988, Theor. Appl. Genet. 76: 835; Christou et al., 1991, Bio/Technology 9: 957); rye (Secale cereale; De la Pena et al., 1987, Nature 325: 274); sorghum (Sorghum bicolor; Cassas et al, 1993, Proc. Natl. Acad. Sci. USA 90: 11212); sugar cane (Saccharum spp.; Bower and Birch, 1992, Plant J. 2: 409); tall fescue (Festuca arundinacea; Wang et al, 1992, Bio/Technology 10: 691); turfgrass (Agrostis palustris; Zhong et al, 1993, Plant Cell Rep. 13: 1); wheat (Triticum aestivum; Vasil et al, 1992, Bio/Technology 10: 667; Troy Weeks et al, 1993, Plant Physiol. 102: 1077; Becker et al, 1994, Plant J. 5: 299).

Methods for transforming and transfecting a wide variety of different dicots and obtaining transgenic plants are well documented in the literature (see Gasser and Fraley, 1989, Science 244: 1293; Fisk and Dandekar, 1993, Scientia Horticulturae, 55: 5-36; Christou, 1994, Agro Food Industry Hi Tech (March/April 1994) p.17, and the references cited therein) and include for example electroporation, particle bombardment, silica fibres and PEG (polyethylene glycol)-mediated transfection, and can also be applied in the present invention. A DNA encoding the expression product of the present invention can be introduced into any of these dicotyledonous plants in order to produce transgenic plants that display dwarf phenotypes in the field.

Plants may be agronomic crop plants, horticultural crop plants or ornamental crop plants. Agronomic and horticultural crop plants include cereals, non- cereal seed crops, root crops, vegetable crops and fruit crops. Cereal crops include wheat, rye, barley, oats, maize, buckwheat, sorghum, and rice; non-cereal seed crops include peas, beans, soybeans, oil-seed rape, canola, linseed, sunflower, and flax; root crops include potato, sweet potato, sugar beet, carrot, swede, and turnip; vegetable crops include asparagus, mustard, lettuce, tobacco, and cauliflower; horticultural crops include tomato, egg plant, cucumber, celery, melon, and squash; fruit crops include strawberry, blackberry, blueberry, apple, apricot, peach, pear, plum, orange, cranberry, and lemon. Additional crop plants include cotton and sugarcane. Ornamental plants include petunia, chrysanthemum, carnation, poinsettia, begonia, tradescantia and snapdragon.

Also provided according to the present invention is a method of inducing a dwarf phenotype in a plant comprising the steps of:

a) transforming or transfecting cells of a plant with a recombinant DNA construct according to the present invention;

b) selecting plant cells that have been transformed or transfected; c) regenerating plant cells that have been transformed or transfected to produce differentiated plants; and

d) selecting a transformed plant which expresses said construct and which has a dwarf phenotype.

Also provided is a plant produced according to the method of the present invention.

Also provided according to the present invention is seed obtained by growing a plant according to the present invention.

The invention will be further apparent from the following description, with reference to accompanying Figure which shows, by way of example only, results obtained with primary transformants.

Experimental

The following experiments detail the isolation, purification and cloning of RNA encoded by gene mybntl and the isolation and sequencing of mybntl from a cDNA library. Transformed tobacco plants expressing mybntl under the control of the CaMV35S promoter were found to have a dominant dwarf phenotype, the dwarfing phenotype being genetically transmissible and the extent of dwarfing being proportional to the level of expression of the mybntl gene.

1. Gene isolation

A member of the myb gene familiy was isolated from a cDNA library prepared from polyA+ RNA isolated from mature pollen of tobacco (Nicotiana tabacum cv. Samsun NN). cDNA library was prepared in the vector lambda ZAP using the techniques of Sambrook, J., Frisch, E.F., and Maniatis, T. ("Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989). The cDNA library was screened using degenerate oligonucleotide probes based on the consensus myb DNA binding domain (Jackson, O. et al, 1991, Plant Cell, 3 : 115-125)

A full-length clone (pmybntl) was isolated and fully sequenced (SEQ ID NO: 1).

2. Starting with the mybntl gene, this was modified by the addition of the CaMV35S 5' and 3' terminal sequences (i.e. the promoter and terminator) (Topfer, R. et al, 1987, Nucleic Acids Res., 15: 5890). CaMV35S promoters and terminators and cloning vectors are commercially available, and include promoters having duplicate enhancer regions at approximately -90 to -343. The CaMV35S5'-mybntl-CaMV35S3' was then modified by the addition of terminal nucleotides to give the fragment Sall- CaMV35S5'-myb-CaMV35S3'-SacI, which was then inserted into plasmid pBIN19 (Bevan, M. et al., 1984, Nucleic Acids Res., 12: 8711-8721) at restriction sites between nos3' and LB to give plasmid pBIN19. This was then introduced into the disarmed Agrobacterium tumefaciens strain LBA4404(pAL4404) (Bevan, M. et al, 1984, supra). This Agrobacterium strain is referred to herein as agrobacterium dwarf (ADW)

3. The Agrobacterium strain ADW was used to infect leaf discs of tobacco (Nicotiana tabacum cv SRI) and kanamycin resistant, stably transformed plants regenerated. Primary transformants showed a reduction in mature plant height (see Figure) referred to as a dwarf phenotype.

4. Plants showing the dwarfing phenotype were allowed to set seed and shown to segregate approximately 3: 1 for kanamycin resistance:sensitivity, consistent with the presence of the T-DNA at a single genetic locus. All kanamycin resistant progeny showed the dwarf phenotype and two classes of dwarf plants were identified, one being severely dwarfed and the other showing intermediate plant height relative to the wild type. Seed collected from the severely dwarfed plants gave rise to 100% kanamycin-resistant progeny and therefore were homozygous for the T-DNA. Plants with intermediate level of dwarfing segregated 3: 1 for kanamycin resistance and were therefore heterozygous for the T-DNA. These data demonstrate that the introduced T-DNA carrying the 35S-mybntl gene fusion can cause a dominant or gene-dosage dependent dwarfing phenotype.

5. The dwarfing phenotype was shown to be genetically transmissible and the severity of dwarfing was positively correlated with the level of expression of the introduced mybntl gene in the transgenic plants.

6. Transcript levels of mybNtlin transgenic plants containing the 35S-mybNtl construct were determined using Northern blot analysis. Northern blotting was performed using standard methodology. Total RNA was isolated from wild-type, myb hemizygous and corresponding homozygous plants and analysed using Northern blot analysis, the Northern blot being probed by incubating it with a radiolabelled mybntl coding sequence and then autoradiographing, RNA transcripts havign the mybntl sequence appearing asdark bands on the autoradiograph.

The results of Northern blotting showed that dwarf plants homozygous for the transgene show higher (appox. 2 fold) levels of expression than heterozygous plants (strictly speaking hemizyous). Plants without the transgene or those which did not show dwarfing did not showdetectable expression of mybNtl .

It was concluded that that the mybNtl over-expression level is positively correlatedwith the degree of dwarfing. This was confirmed in several independent transgenic lines.

7. Additional height data:

The height of plants homozygous and hemizygous for the mybNtl transgene construct was examined. Results show that for several independent transgenic lines that the homozygous plants are significantly more dwarfed than the heterozygotes.

From the above data it was conclude that gene dosage of the mybntl construct in plants is positively correlated with the degree of dwarfing.

Taken together with the results of Northern blotting, these results establish that mybNtl overexpression using the 35S promoter (and in this example in tobacco) results in dwarfing that is dependent upon gene dosage acting through expression level.

Therefore tissue-targeted and inducible control of plant and organ growth can be expected to be possible using such an expression-dependent strategy.

Claims

1. A plant gene having the sequence of SEQ ID NO: 1

2. The expression product of a plant gene according to claim 1.

3. A recombinant DNA construct whose expression in plants induces a dwarf phenotype. and comprising operatively linked in sequence in the 5' to 3' direction:

a) a promoter region that directs the transcription of a gene in plants;

b) a DNA coding sequence encoding an RNA sequence encoding an expression product according to claim 2; and

c) a 3' non-translated region which contains a polyadenylation signal for plants.

4. A recombinant DNA construct according to claim 2, the expression of the construct being in plant tissues other than pollen.

5. A recombinant DNA construct according to either one of claims 3 or 4, the 3' non-translated region encoding a polyadenylation signal which functions in plant cells to cause transcription termination and the addition of polyadenylate nucleotides to the 3' end of the RNA sequence.

6. A recombinant DNA construct according to any one of claims 3-5, the DNA coding sequence having the sequence of SEQ ID NO: 1.

7. A recombinant DNA construct according to any one of claims 3-6, the promoter comprising the CaMV35S promoter.

8. A recombinant DNA construct according to any one of claims 3-7, the 3' non-translated region comprising the 3' polyadenylation signal from the CaMV35S transcript.

9. A plant transformed or transfected with a recombinant DNA construct according to any one of claims 3-8.

10. A method of inducing a dwarf phenotype in a plant comprising the steps of:

a) transforming or transfecting cells of a plant with a recombinant DNA construct according to any one of claims 3-8;

b) selecting plant cells that have been transformed or transfected;

c) regenerating plant cells that have been transformed or transfected to produce differentiated plants; and

d) selecting a transformed plant which expresses said construct and which has a dwarf phenotype.

11. A plant produced according to the method of claim 10.

12. Seed obtained by growing a plant according to either one of claims 9 or 11.